title: Transcriptional repression in the Notch pathway: thermodynamic characterization of CSL-MINT (Msx2-interacting nuclear target protein) complexes.
abstract: The Notch pathway is a conserved cell-to-cell signaling mechanism that mediates cell fate decisions in metazoans. Canonical signaling results in changes in gene expression, which is regulated by the nuclear effector of the pathway CSL (CBF1/RBP-J, Su(H), Lag-1). CSL is a DNA binding protein that functions as either a repressor or an activator of transcription, depending upon whether it is complexed by transcriptional corepressor or coactivator proteins, respectively. In stark contrast to CSL-coactivator complexes, e.g. the transcriptionally active CSL-Notch-Mastermind ternary complex, the structure and function of CSL-corepressor complexes are poorly understood. The corepressor MINT (Msx2-interacting nuclear target protein) has been shown in vivo to antagonize Notch signaling and shown in vitro to biochemically interact with CSL; however, the molecular details of this interaction are only partially defined. Here, we provide a quantitative thermodynamic binding analysis of CSL-MINT complexes. Using isothermal titration calorimetry, we demonstrate that MINT forms a high affinity complex with CSL, and we also delineate the domains of MINT and CSL that are necessary and sufficient for complex formation. Moreover, we show in cultured cells that this region of MINT can inhibit Notch signaling in transcriptional reporter assays. Taken together, our results provide functional insights into how CSL is converted from a repressor to an activator of transcription.
authors.affiliation: Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267, USA.
authors.forename: Bradley D
authors.initials: BD
authors.lastname: VanderWielen
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Yuan
authors.forename: David R
authors.initials: DR
authors.lastname: Friedmann
authors.forename: Rhett A
authors.initials: RA
authors.lastname: Kovall
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology; D005786:Gene Expression Regulation; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D016601:RNA-Binding Proteins; D051880:Receptors, Notch / Q000037:antagonists & inhibitors / Q000235:genetics*; D012097:Repressor Proteins; D013816:Thermodynamics; D015534:Trans-Activators; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins; D051819:Immunoglobulin J Recombination Signal Sequence-Binding Protein; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C497571:Rbpj protein, mouse; D051880:Receptors, Notch; D012097:Repressor Proteins; C121253:Spen protein, mouse; D015534:Trans-Activators